1. Field of the Invention
This invention relates generally to compositions and methods of propagating hepatitis C viral genomes and infectious particles. Specifically, the invention is directed to an immortalized human hepatic cell line comprising the hepatitis C viral 1a genome, (IHH/HCV 1a) which is capable of replicating the hepatitis C viral 1a (HCV 1a) genotype and producing fully infectious HCV 1a virus particles.
2. Description of the Related Art
According to the American Liver Foundation, over 300,000 Americans are hospitalized each year for cirrhosis of the liver. The primary causes of cirrhosis are alcohol abuse and chronic hepatitis C(HCV). To date, approximately 3.9 million Americans suffer from hepatitis C. The most important feature of HCV infection is the development of chronic hepatitis in a significant number of infected individuals and the potential for disease progression to cirrhosis and hepatocellular carcinoma (6, 7, 11, 27). At present, the only approved therapy for chronic HCV infection is interferon (IFN)-α with or without ribavirin (9, 21), but this therapy fails to clear HCV from a significant number of patients (22). A number of HCV genomes have been cloned, and sequence divergence indicates several genotypes as well as a series of subtypes for this virus (28). In the United States, HCV genotypes 1a and 1b are predominant in patients with chronic hepatitis C (31). Progress in the understanding HCV biology has been hampered due to the lack of an efficient cell culture system for virus growth. The establishment of self-replicating HCV full-length genomic replicons from genotypes 1a and 1b in human hepatoma (Huh-7) cells has provided an important tool for the study of HCV replication mechanisms (3, 10, 23). Although some groups have reported the generation of infectious virus from transfection of genomic RNA of HCV genotype 2a into Huh-7 (5, 15, 29, 32), the generation of infectious HCV genotype 1a has not been successful to date, and therefore a long felt need exists.
The inventors and others have previously shown that HCV core protein transcriptionally regulates a number of cellular genes (26). The inventors have also previously described the generation of immortalized human hepatocytes (IHH) by transfection of the HCV core genomic region from genotype 1a (2, 25). IHH exhibit a weak level of HCV core protein expression, albumin secretion, glucose phosphatase activity, and absence of smooth muscle actin. IHH also displayed focal cytoplasmic and membrane staining for carcinoembryonic antigen (CEA), biliary glycoprotein (BGP1/CEACAM1) and nonspecific cross-reacting antigen (NCA/CEACAM6), and expression of hepato-biliary transport marker genes (MRP, LST1 and NTCP). Together, these results suggested that IHH are well differentiated. HCV core protein selectively degrades STAT1, reduces phosphorylated STAT1 (P-STAT1) accumulation in the nucleus in a proteasome-dependent manner, and impairs IFN-α-induced signal transduction via suppressor of cytokine signaling-3 expression (1, 4, 16). HCV core protein is competent to partially rescue growth of a genetically engineered influenza A virus lacking its own IFN antagonist (4). The core protein can modulate interferon regulatory factor (IRF), Jak-STAT and inducible nitric oxide synthetase (iNOS) pathways, and suggest mechanisms by which core could affect HCV persistence and pathogenesis (20). Since HCV core protein transcriptionally regulates several cellular genes involved in cell growth, apoptosis and defense mechanism, the inventors hypothesize that IHH may set the stage for HCV genome replication and assembly.
The inventors have sought to address the long felt need of providing a cell line permissible for HCV 1a replication and generation of infections virus particles. A cell line with this capability will be invaluable to researchers not only by providing easy access to HCV 1a infectious virus particles, but to identify HCV mediators and their pathways. In addition, it will be invaluable as a tool to screen new therapeutic strategies or potential pharmacological agents which interfere with the propagation of HCV and resulting diseases caused by the virus.